There is significant need for effective antimycotic drugs especially for the treatment of systemic fungal infections which are life-threatening, common complications in immune-compromised patients, see for example Hart et al. (1969) J. Infect. Dis. 120:169-191. Among the most virulent organisms are strains of the yeast Candida, most particularly strains of C. albicans. While there are several effective topical agents for treatment of candidiases, treatment of systemic infection is much more difficult. The drug of choice for systemic infection is amphotericin B, however this drug is highly toxic to the host (see, for example, Medoff and Kobayashi (1980) New Eng. J. Med. 302:145-55). Antimycotic agents that are more effective and/or less toxic than existing drugs are highly desirable.
Several classes of nucleoside antibiotics, including polyoxins (Hori et al. (1971) Agr. Biol. Chem. 35:1280; Hori et al. (1974) Agr. Biol. Chem. 38:699; Sasaki et al. (1968) Ann. Phytopathol. Soc. Japan 34:272) and nikkomycins (Dahn et al. U.S. Pat. Nos. 4,046,881 and 4,158,608; Zahner et al. U.S. Pat. No. 4,287,186; Hagenmaier et al. U.S. Pat. No. 4,315,922) have been reported. Polyoxins and nikkomycins are reported to be useful in agriculture against phytopathogenic fungi and insect pests. Early reports indicated that polyoxins were not effective against zoopathogenic fungi, such as C. albicans (see, for example, Gooday (1977) J. Gen. Microbiol. 99:1; Shenbagamurthi et al. (1983) J. Med. Chem. 26:1518-1522). It was believed that the polyoxins were not taken up by target cells. More recently, polyoxins have been reported to inhibit the growth in vitro of certain zoopathogenic fungi including C. albicans and Cryptococcus neoformans when provided at millimolar concentrations (Becker et al. Antimicro. Agents Chemother. (1983) 23:92-929 and Mehta et al. (1984) Antimicro. Agents Chemother. 25:373-374). Nikkomycins X and Z have now also been reported to inhibit growth of C. albicans in vitro (Yadan et al. (1984) J. Bacteriol. 160:884-888; McCarthy et al. (1985) J. Gen. Microbiol. 131:775-780). Polyoxins and nikkomycins are similar in structure and apparently both act as competitive inhibitors of chitin synthetase (Endo et al. (1970) J. Bacteriol. 104:189-196; Muller et al. (1981) Arch. Microbiol. 130:195-197). Chitin is an essential component of the cell wall of most fungi. Nikkomycins appear, however, to be more effective (about 100 fold) against certain fungi, for example C. albicans, than polyoxins which is in part due to a higher affinity of nikkomycin for chitin synthetase and more rapid uptake of nikkomycin by C. albicans cells (McCarthy et al. (1985) supra). The activity of polyoxins and nikkomycins is reported to be inhibited by peptides, such as those present in rich media (Becker et al., 1983, supra; McCarthy et al., 1985, supra Mehta et al., 1984, supra). Peptides are believed to inhibit uptake of the antibiotic by target cells. The usefulness of nikkomycins and polyoxins for clinical applications such as in the treatment of systemic fungal infection, where peptide inhibition is likely, is expected to be limited as the concentrations of antibiotic required for effective fungal inhibition are not likely to be achieved in vivo.
Mixtures of antimicrobial agents, particularly mixtures in which the components have different modes of action have been used in antimicrobial compositions to broaden activity spectrum or to minimize the occurrence of resistant strains. Further, certain of these mixtures can display an enhanced antimicrobial activity, greater than the additive activity of the individual components, due to synergy. For example, Gisi et al. (1985) Trans. Br. Mycol. Soc. 85:299-306 reported that a number of fungicide mixtures displayed synergistic activity against phytopathogenic fungi in field tests. The maximum synergy ratio reported was 7, that is a 7-fold enhancement of activity over the calculated additive effect. Fungicide mixtures can also show antagonism with reduced activity of the combination compared to the individual components. It has recently been reported (Hector and Braun (1986) Antimicro. Agent Chemother. 29:389-394) that mixtures of either nikkomycin Z or nikkomycin X with papulacandin B, an inhibitor of .beta.-glucan synthesis, display synergistic antifungal activity against Candida albicans. Activity enhancements up to about 10 were reported.
Certain enzymes have also been reported to synergize the effect of antifungal agents. Lysozyme has been reported to synergize the activity of amphotericin B against Candida albicans and Coccidioides immitis (Collins and Pappagianis (1974) Sabouraudia 12:329-340). Natural mixtures of mycolytic enzymes of fungal origin, designated mycolases, were reported to have a synergistic effect on the activity of the antifungal drugs amphotericin B and nystatin (Davies and Pope (1978) Nature 273:235-6; Pope and Davies (1979) Postgraduate Med. J. 55:674-676). The in vitro MICs (minimum inhibitory concentrations) of these antifungal drugs were lowered about 5 to 10-fold in combinations with mycolase. In related in vivo experiments in a mouse model, fungal mycolase was reported to enhance the effectiveness of amphotericin B and nystatin against systemic infection of C. albicans. It was suggested that mycolase, which was suggested to be a mixture of carbohydrases, enhanced penetration of the antibiotic into fungal cells. Fungal mycolases, alone, were described as very effective at releasing protoplasts from Aspergillus fumigatus and C. albicans in vitro and were also reported to have some effect, alone, against systemic fungal infection in the mouse model system. In contrast, a prepared mixture of the carbohydrases chitinase (.beta.-1,4 N-acetyl-D-glucosaminidase) and laminarinase (.beta.-1,3(4)-glucanase), while reported to effect protoplast release from A. fumigatus and C. albicans, did not enhance the effectiveness of amphotericin B and nystatin in vivo. Recently, in similar in vitro and in vivo experiments with fungal mycolase/amphotericin B mixtures, only slight enhancement of antifungal activity by a fungal mycolase was reported (Chalkley et al. (1985) Sabouraudia 23:147-164). This report suggests that the difference in results compared to those reported earlier by Davies and Pope (supra) may be associated with the lower chitinase or lower .beta.-1,6-D-glucanase activities in their preparation of mycolase compared to that employed in the previous experiments. The specific enzymatic activities present in fungal mycolases have not been identified, and the specific protein or proteins in mycolase that may effect antibiotic enhancement have not been identified Some bacterial mycolases have also been reported to effect enhancements (about 2-fold) of the activity of amphotericin B (Oranusi and Trinci (1985) Microbios 43:17-30). Again, no specific enzyme activity was associated with synergy.
Plants appear to have a variety of mechanisms for protecting themselves against infection by viruses, bacteria, fungi and insects. These mechanisms are believed to include the presence of inhibitory substances in plant tissue or plant excretions. Such inhibitory substances may be present constitutively in the plant or induced by infection and may be low molecular weight compounds such as inhibitins or phytoalexins or certain proteins, for example, peroxidases, proteinase inhibitors, chitinases or .beta.-1,3-glucanases. In most cases, the inhibitory function of these substances have not been demonstrated.
In addition to the specific need for more effective, clinically useful antifungal agents, there is a general need for effective, natural, biodegradable antifungal agents, particularly for use in agriculture against plant pathogenic fungi. Such natural antifungal agents may be considered to be ecologically preferable to chemical fungicides. To be economically useful, especially in agricultural applications, such natural antifungal agents should be available in large amounts from inexpensive sources.
It has been recently reported, Roberts and Selitrennikoff (1986) Biochem. Biophys. Acta 880:161-170, that a class of plant proteins called ribosome-inactivating proteins (RIPs) are effective against certain fungi, for example, Trichoderma reesei. These proteins were earlier shown to inhibit protein synthesis in animal cell-free extras (Coleman and Roberts (1982) Biochem. Biophys. Acta 696:239-244). RIPs isolated from grains, including wheat, barley, rye and corn, reportedly act by enzymatically inactivating the 60S subunit of the animal cell ribosome. Coleman and Roberts (1982) reported that RIPs could be purified to near homogeneity from rye, barley, corn and tritin using the same procedure (roberts and Stewart (1979) Biochemistry 18:2615-2621). RIPs from rye, barley and tritin were reported to have apparent molecular weights of approximately 30 kd when analyzed by SDS-PAGE run under reducing conditions, and corn RIP was reported to run as an approximately 23 kd protein under similar conditions. Roberts and Selitrennikoff (1986) supra demonstrated that RIP isolated from barley inactivated Neurospora ribosomes as measured by in vitro inhibition of poly(U)-directed polyphenylalanine synthesis in cytoplasmic ribosome preparations (see also Coleman and Roberts (1981) Biochim. Biophys. Acta 659:57-66). These authors have also reported the presence in barley, corn, wheat and rye of another class of antifungal proteins, designated AFPs which inhibit growth of some fungi, including T. reesei, in vitro. The present work is an extension of this work with plant antifungal proteins.